Maintenance of differentiated cells with laminins

ABSTRACT

The present disclosure describes methods of maintaining the phenotype of differentiated cells. Generally, the natural environment of the body is replicated for the differentiated cell. The differentiated cell is plated on a cell culture substrate comprising a laminin, such as laminin-521 or laminin-511. The substrate may also contain a cadherin. This maintains the phenotype of the differentiated cell.

This application is a continuation of U.S. patent application Ser. No.15/351,124, filed on Nov. 14, 2016, now U.S. Pat. No. 11,155,781, whichis a continuation of U.S. patent application Ser. No. 13/866,177, filedon Apr. 19, 2013, now U.S. Pat. No. 9,499,794, which claims priority toU.S. Provisional Patent Application Ser. No. 61/716,005, filed on Oct.19, 2012; to U.S. Provisional Patent Application Ser. No. 61/754,784,filed on Jan. 21, 2013; to U.S. Provisional Patent Application Ser. No.61/636,293, filed on Apr. 20, 2012; and to U.S. Provisional PatentApplication Ser. No. 61/636,211, filed on Apr. 20, 2012. The disclosureof each of these applications is hereby fully incorporated by reference.

BACKGROUND

A stem cell is an undifferentiated cell from which specialized cells aresubsequently derived. Examples of stem cells in the human body includepluripotent stem cells, embryonic stem cells, adult stem cells, fetalstem cells, and amniotic stem cells. Embryonic stem cells possessextensive self-renewal capacity and pluripotency with the potential todifferentiate into cells of all three germ layers.

Totipotency refers to a cell that has the ability to differentiate intoany cell in the body, including extraembryonic tissue. Pluripotencyrefers to a cell that has the potential to differentiate into cells ofall three germ layers. Pluripotent cells however cannot formextraembryonic tissue, as a totipotent cell can. Multipotency refers toa cell that can differentiate into cells of limited lineage. Forexample, a hematopoietic stem cell can differentiate into several typesof blood cells, but cannot differentiate into a brain cell.

The process by which a stem cell changes into a more specialized cell isreferred to as differentiation. For example, some differentiated cellsinclude endothelial cells, which are derived from endothelial stemcells.

The process by which a specialized cell reverts back to a higher degreeof potency (i.e. to an earlier developmental stage) is referred to asdedifferentiation. In particular, cells in a cell culture can loseproperties they originally had, such as protein expression or shape. Itwould be desirable to reduce the rate of dedifferentiation, or in otherwords to maintain the phenotype of differentiated cells in a cellculture.

BRIEF DESCRIPTION

Disclosed herein are methods for maintaining the phenotype ofdifferentiated cells in a cell culture.

Described herein are methods for maintaining the phenotype of adifferentiated cell, comprising: plating the differentiated cell on acell culture substrate comprising a laminin, wherein the laminin is anintact protein or a protein fragment.

The differentiated cell can be an endothelial cell, a cardiomyocyte, adopamine producing cell, a hepatocyte, or a pancreatic beta cell.

The laminin may be laminin-521 or laminin-511, or an effectiverecombinant laminin.

The cell culture substrate may further comprise a cadherin. Sometimes,the cadherin is e-cadherin. The weight ratio of the laminin to thecadherin can be from about 5:1 to about 15:1, or from about 5:1 to about10:1. In particular embodiments, the laminin is laminin-521 and thecadherin is e-cadherin. In other embodiments, the cell culture substrateconsists of the laminin and the cadherin. Generally, the cell culturesubstrate does not contain any differentiation inhibitors, feeder cells,differentiation inductors, or apoptosis inhibitors.

The method may further include applying a cell culture medium to thefirst stem cell. In specific embodiments, the cell culture medium has analbumin concentration of at least 0.3 mM.

These and other non-limiting characteristics of the disclosure are moreparticularly disclosed below.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following is a brief description of the drawings, which arepresented for the purposes of illustrating the exemplary embodimentsdisclosed herein and not for the purposes of limiting the same.

FIG. 1 is a rotary shadowing electron microscopy picture of arecombinant laminin molecule.

FIG. 2 shows the structural motifs of laminin α, β, and γ chains. TheN-terminal, internal, and C-terminal globular domains are depicted aswhite ovals. The coiled-coil forming domains (I and II) are shown aswhite rectangles. The rod-like structures (domains V, IIIb, and IIIa)are depicted as grey rectangles.

FIG. 3 is a photomicrograph of human umbilical vein endothelial cells(HUVECs) grown on a fibronectin (FNE) substrate, 10× magnification,after 5 passages, with expression of von Willebrand factor (vWF),f-actin, and DAPI.

FIG. 4 is a photomicrograph of HUVECs grown on a LN-521 substrate, 10×magnification, after 5 passages, with expression of vWF, f-actin, andDAPI.

FIG. 5 is a photomicrograph of HUVECs grown on a LN-521 substrate, 10×magnification, after 7 passages, with expression of vWF, f-actin, andDAPI.

FIG. 6 is a photomicrograph of HUVECs grown on a LN-411/511 substrate,10× magnification, after 5 passages, with expression of vWF, f-actin,and DAPI.

FIG. 7 is a photomicrograph of HUVECs grown on a LN-511 substrate, 10×magnification, after 5 passages, with expression of vWF, f-actin, andDAPI.

FIG. 8 is graph of RNA Gene Expression showing relatively low Acta2 geneexpression (negative marker) (the five leftmost columns) and high vWFgene expression (positive marker) (the five rightmost columns) relativeto a control (Fibronectin) when cells are grown on a LN-511 substrate.

FIG. 9 is a graph of quantified percentage of vWF-positive HUVECs withina population after a long-term culture of HUVECs on human recombinantlaminin-521.

FIG. 10 is graph of quantified percentage of vWF-positive HUVECs withina population after a long-term culture of HUVECs on human recombinantFibronectin.

FIG. 11 is proliferation curve showing the proliferation of HUVECs ondifferent substrate coatings, dependent of days in culture. The LN-521line always has the greatest value. The LN-411/511 and LN-511 linesessentially overlap until −115 days, at which point the LN-511 line isgreater. The FNE line has the lowest value until −80 days, when it thencrosses over to have the second-highest value.

FIG. 12 is a phase-contrast micrograph of mouse pancreaticinsulin-producing islet beta cells plated on a surface coated withLN-521, 10× magnification, 3 week culture.

FIG. 13 is a phase-contrast micrograph of mouse pancreaticinsulin-producing islet beta cells plated on an uncoated surface, 10×magnification, 3 week culture.

FIG. 14 is a phase-contrast micrograph of mouse pancreaticinsulin-producing islet beta cells plated on a surface coated withLN-411, 10× magnification, 3 week culture.

FIG. 15 is a phase-contrast micrograph of mouse pancreaticinsulin-producing islet beta cells plated on a surface coated withLN-511, 10× magnification, 3 week culture.

FIG. 16 is a phase-contrast micrograph of mouse pancreaticinsulin-producing islet beta cells plated on a surface coated withLN-111, 10× magnification, 3 week culture.

FIG. 17 is a phase-contrast micrograph of mouse pancreaticinsulin-producing islet beta cells plated on a surface coated with humanrecombinant LN-521 at (a) 10× magnification and (b) 40× magnification.

FIG. 18 is a phase-contrast micrograph of mouse pancreaticinsulin-producing islet beta cells plated on a surface coated with humanrecombinant LN-111 at (a) 10× magnification and (b) 40× magnification.

FIG. 19 is a phase-contrast micrograph of mouse pancreaticinsulin-producing islet beta cells plated on a surface coated with humanrecombinant LN-521 for 3-4 weeks at 10× magnification and subsequently(a) stained positively for C-peptide (green) and (b) stained positivelyfor C-peptide (green) and Hoechst (blue).

FIG. 20 is a phase-contrast micrograph of mouse pancreaticinsulin-producing islet beta cells plated on a surface coated with humanrecombinant LN-521 for 3-4 weeks at 20× magnification and subsequently(a) stained positively for EdU (green) in nuclei of proliferated cellsand (b) stained positively for Edu (green) co-localized with phasecontrast photograph of islet.

FIG. 21 is a phase-contrast micrograph of mouse pancreaticinsulin-producing islet beta cells plated on a surface coated with humanrecombinant LN-521 for 3-4 weeks at 20× magnification and subsequently(a) stained positively for EdU (green) in nuclei of proliferated cellsand (b) stained positively for Edu (green) co-localized with Hoechst(blue).

DETAILED DESCRIPTION

A more complete understanding of the compositions and methods disclosedherein can be obtained by reference to the accompanying drawings. Thesefigures are merely schematic representations based on convenience andthe ease of demonstrating the present disclosure, and are, therefore,not intended to define or limit the scope of the exemplary embodiments.

Although specific terms are used in the following description for thesake of clarity, these terms are intended to refer only to theparticular structure of the embodiments selected for illustration in thedrawings, and are not intended to define or limit the scope of thedisclosure. In the drawings and the following description below, it isto be understood that like numeric designations refer to components oflike function.

All publications, patents, and patent applications discussed herein arehereby incorporated by reference in their entirety.

Unless otherwise stated, the techniques utilized in this application maybe found in any of several well-known references such as: MolecularCloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring HarborLaboratory Press), Gene Expression Technology (Methods in Enzymology,Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego,Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P.Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide toMethods and Applications (Innis, et al. 1990. Academic Press, San Diego,Calif.), Culture of Animal Cells: A Manual of Basic Technique, SecondEd. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N.J.), or the Ambion 1998 Catalog (Ambion, Austin, Tex.).

The methods of the present disclosure are generally related tomaintaining the phenotype of differentiated cells. The term “phenotype”here refers to the cell's observable characteristics and properties.These include such things as the cell's morphology, biochemical orphysiological properties, etc. It is desirable to maintain the cell'sphenotype.

It is contemplated that any kind of differentiated cell can bemaintained with the methods of the present disclosure. Examples ofdifferentiated cells include endothelial cells, cardiomyocytes, dopamineproducing cells, hepatocytes, and pancreatic beta cells, though ofcourse other differentiated cells are contemplated. Generally speaking,the present disclosure creates a natural environment for thedifferentiated cell using laminins that are close to the differentiatedcell in the body.

The methods of the present disclosure also relate to improving thetransfection efficiency of primary cells and/or survival rate oftransfected cells. The primary cells are plated on a substratecontaining a laminin, wherein the laminin is an intact protein or aprotein fragment. The primary cells are then transfected with a vector,and the transfected cells are cultured on the substrate.

The term “primary cells” refers in the art to cells which are takendirectly from a subject. Such cells generally are not immortal, and havea limited lifespan, or in other words they stop dividing though theyretain viability. Exemplary primary cells include hepatocytes,adipocytes, podocytes, chondrocytes, melanocytes, keratinocytes, andlaminins. Primary cells are also differentiated cells.

Differentiated cells typically require two things to survive andreproduce: (1) a substrate or coating that provides a structural supportfor the cell; and (2) a cell culture medium to provide nutrition to thecell. The substrate or coating (1) is typically formed as a layer in acontainer, for example a petri dish or in the well of a multi-wellplate. It is particularly contemplated that the cell culture substrateon which the differentiated cell is plated comprises a laminin and acadherin.

Laminins are a family of heterotrimeric glycoproteins that resideprimarily in the basal lamina. They function via binding interactionswith neighboring cell receptors on the one side, and by binding to otherlaminin molecules or other matrix proteins such as collagens, nidogensor proteoglycans. The laminin molecules are also important signalingmolecules that can strongly influence cellular behavior and function.Laminins are important in both maintaining cell/tissue phenotype, aswell as in promoting cell growth and differentiation in tissue repairand development.

Laminins are large, multi-domain proteins, with a common structuralorganization. The laminin molecule integrates various matrix and cellinteractive functions into one molecule.

A laminin protein molecule comprises one α-chain subunit, one β-chainsubunit, and one γ-chain subunit, all joined together in a trimerthrough a coiled-coil domain. FIG. 1 depicts the resulting structure ofthe laminin molecule. The twelve known laminin subunit chains can format least 15 trimeric laminin types in native tissues. Within thetrimeric laminin structures are identifiable domains that possessbinding activity towards other laminin and basal lamina molecules, andmembrane-bound receptors. FIG. 2 shows the three laminin chain subunitsseparately. For example, domains VI, IVb, and IVa form globularstructures, and domains V, IIIb, and IIIa (which contain cysteine-richEGF-like elements) form rod-like structures. Domains I and II of thethree chains participate in the formation of a triple-strandedcoiled-coil structure (the long arm).

There exist five different alpha chains, three beta chains and threegamma chains that in human tissues have been found in at least fifteendifferent combinations. These molecules are termed laminin-1 tolaminin-15 based on their historical discovery, but an alternativenomenclature describes the isoforms based on their chain composition,e.g. laminin-111 (laminin-1) that contains alpha-1, beta-1 and gamma-1chains. Four structurally defined family groups of laminins have beenidentified. The first group of five identified laminin molecules allshare the β1 and γ1 chains, and vary by their α-chain composition (α1 toα5 chain). The second group of five identified laminin molecules,including laminin-521, all share the β2 and γ1 chain, and again vary bytheir α-chain composition. The third group of identified lamininmolecules has one identified member, laminin-332, with a chaincomposition of α3β3γ2. The fourth group of identified laminin moleculeshas one identified member, laminin-213, with the newly identified γ3chain (α2β1γ3).

Generally, the cell culture substrate may contain any effective laminin,wherein the effectiveness is determined by whether differentiated cellscan survive upon the substrate. It is specifically contemplated that thesubstrate contains only one particular laminin, though other ingredientsare also present in the substrate. In specific embodiments, the lamininis laminin-521 (LN-521) or laminin-511 (LN-511).

The term “laminin-521” refers to the protein formed by joining α5, β2and γ1 chains together. The term “laminin-511” refers to the proteinformed by joining α5, β1 and γ1 chains together. These terms should beconstrued as encompassing both the recombinant laminin andheterotrimeric laminin from naturally occurring sources. The term“recombinant” indicates that the protein is artificially produced incells that do not normally express such proteins.

The laminin can be an intact protein or a protein fragment. The term“intact” refers to the protein being composed of all of the domains ofthe α-chain, β-chain, and γ-chain, with the three chains being joinedtogether to form the heterotrimeric structure. The protein is not brokendown into separate chains, fragments, or functional domains. The term“chain” refers to the entirety of the alpha, beta, or gamma chain of thelaminin protein. The term “fragment” refers to any protein fragmentwhich contains one, two, or three functional domains that possessesbinding activity to another molecule or receptor. However, a chainshould not be considered a fragment because each chain possesses morethan three such domains. Similarly, an intact laminin protein should notbe considered a fragment. Examples of functional domains include DomainsI, II, III, IV, V, VI, and the G domain.

The average contact area and spreading homogeneity is much larger forcells cultured on laminin-511 compared to other available substrata.

In particular, it is noted that the pancreatic insulin-producing isletsare naturally in the shape of a three-dimensional sphere. However, apetri dish typically only provides two dimensions for growth, whichmeans that it is difficult to expand the islets using a mechanicalsplit. Beta cells within the islets form syncytium-like structures, andbeta cells will respond simultaneously to external signals. Whencultured as single cells, though, beta cells lose this natural functionof simultaneous response.

The cell culture substrate also comprises a cadherin. Cadherins are aclass of type-1 transmembrane proteins that play important roles in celladhesion, ensuring that cells within tissues are bound together. Theyare dependent on calcium (Ca²⁺) ions to function. Cadherins are alsoknown as desmogleins and desmocollins. Structurally, cadherins containextracellular Ca²⁺-binding domains. In particular embodiments, thecadherin used in the cell culture substrate is epithelial cadherin ore-cadherin.

The weight ratio of the laminin to the cadherin may be from about 5:1 toabout 15:1, or from about 5:1 to about 10:1. In particular embodiments,the cell culture substrate consists of the laminin and the cadherin. Inother specific embodiments, the laminin is laminin-521 and the cadherinis e-cadherin.

The cell culture substrate is used in combination with a cell culturemedium. The cell culture medium of the present disclosure isparticularly suitable for being used with a substrate that containslaminin-521 and/or laminin-511. These laminins activate α6β1 integrins,which in turn leads to activation of the PI3K/Akt pathway. Thisincreases the pluripotency, self-renewal, and/or proliferation of thedifferentiated cells. It is contemplated that the substrate may consistof laminin-521 or laminin-511, either intact, as separate chains, or asfragments thereof. Recombinant laminin-521 and recombinant laminin-511are commercially available; see for example U.S. Pat. No. 8,415,156,which provides amino acid sequences and DNA sequences for LN-521, andthe entirety of which is incorporated by reference herein. Manydifferent molecules can activate the PI3K/Akt pathway, though withdifferent efficiencies. For example, TGF beta 1 and bFGF activate thispathway. The use of laminin-521 and/or laminin-511 allows the quantityof such molecules to be reduced in the cell culture medium. Laminin-521conveys the highest dose of signal via α6β1 integrin, activating thePI3K/Akt pathway. The use of laminin-521 allows for single-cellsuspension passaging without the addition of cell-detrimental rho-kinase(ROCK) inhibitor to increase cell survival after single-cell enzymaticdissociation.

Typically, cell culture media include a large number and a large amountof various growth factors and cytokines to inhibit differentiation andimprove proliferation. One advantage of the cell culture medium of thepresent disclosure is that it does not contain as many growth factors orcytokines, or such high amounts.

Very generally, the cell culture medium of the present disclosurerequires lower amounts of basic fibroblast growth factor (bFGF) thantypically used. It is contemplated that the cell culture medium maycomprise from greater than zero to 3.9 nanograms per milliliter (ng/mL)of bFGF. The bFGF is human bFGF so that the cell culture medium istotally human and defined. In some more specific embodiments, the cellculture medium may comprise 3.5 or lower ng/mL of bFGF. In otherembodiments, the cell culture medium may comprise from 0.5 to 3.5 ng/mLof bFGF. In some embodiments, the cell culture medium may have zerobFGF, i.e. no bFGF is present.

Generally, the cell culture medium includes a liquid phase in which atleast one inorganic salt, at least one trace mineral, at least oneenergy substrate, at least one lipid, at least one amino acid, at leastone vitamin, and at least one growth factor (besides bFGF) aredissolved. Table 1 below includes a list of various such ingredientswhich may be present in the cell culture medium of the presentdisclosure, and the minimum and maximum concentrations if the ingredientis present. The values are presented in scientific notation. Forexample, “4.1E-01” should be interpreted as 4.1×10⁻⁰¹.

TABLE 1 molar Min. Max. Min. Max. mass Conc. Conc. Conc. Conc.Ingredient (g/mol) (mM) (mM) (ng/mL) (ng/mL) INORGANIC SALTS Calciumchloride 110.98 4.1E−01 1.6E+00 4.6E+04 1.8E+05 (Anhydrous) HEPES 238.35.9E+00 1.8E+01 1.4E+06 4.2E+06 Lithium Chloride 42.39 4.9E−01 1.5E+002.1E+04 6.2E+04 (LiCl) Magnesium chloride 95.21 1.2E−01 3.6E−01 1.1E+043.4E+04 (Anhydrous) Magnesium Sulfate 120.37 1.6E−01 4.8E−01 1.9E+045.8E+04 (MgSO₄) Potassium 74.55 1.6E+00 4.9E+00 1.2E+05 3.6E+05 chloride(KCl) Sodium bicarbonate 84.01 9.0E+00 4.4E+01 7.6E+05 3.7E+06 (NaHCO₃)Sodium chloride 58.44 4.7E+01 1.4E+02 2.8E+06 8.3E+06 (NaCl) Sodium141.96 2.0E−01 5.9E−01 2.8E+04 8.3E+04 phosphate, dibasic (Anhydrous)Sodium 137.99 1.8E−01 5.3E−01 2.4E+04 7.3E+04 phosphate, monobasicmonohydrate (NaH₂PO₄E—H₂O) TRACE MINERALS Ferric Nitrate 404 4.9E−051.9E−04 2.0E+01 7.5E+01 (Fe(NO₃)₃—9H₂O) Ferrous sulfate 278.01 5.9E−041.8E−03 1.6E+02 4.9E+02 heptahydrate (FeSO₄—7H₂O) Copper(II) sulfate249.69 2.0E−06 8.0E−06 5.1E−01 2.0E+00 pentahydrate (CuSO₄—5H₂O) Zincsulfate 287.56 5.9E−04 1.8E−03 1.7E+02 5.1E+02 heptahydrate (ZnSO₄—7H₂O)Ammonium 116.98 5.5E−06 1.6E−05 6.4E−01 1.9E+00 Metavanadate NH₄VO₃Manganese Sulfate 169.02 9.9E−07 3.0E−06 1.7E−01 5.0E−01 monohydrate(MnSO₄—H₂O) NiSO₄—6H₂O 262.85 4.9E−07 1.5E−06 1.3E−01 3.8E−01 Selenium78.96 8.9E−05 2.7E−04 7.0E+00 2.1E+01 Sodium Meta 284.2 4.8E−04 1.4E−031.4E+02 4.1E+02 Silicate Na₂SiO₃—9H₂O SnCl₂ 189.62 6.2E−07 1.9E−061.2E−01 3.5E−01 Molybdic Acid, 1235.86 9.9E−07 3.0E−06 1.2E+00 3.7E+00Ammonium salt CdCl₂ 183.32 6.1E−06 1.8E−05 1.1E+00 3.4E+00 CrCl₃ 158.369.9E−07 3.0E−06 1.6E−01 4.7E−01 AgNO₃ 169.87 4.9E−07 1.5E−06 8.3E−022.5E−01 AlCl₃—6H₂O 241.43 2.4E−06 7.3E−06 5.9E−01 1.8E+00 Barium Acetate255.42 4.9E−06 1.5E−05 1.3E+00 3.8E+00 (Ba(C₂H₃O₂)₂) CoCl₂—6H₂O 237.934.9E−06 1.5E−05 1.2E+00 3.5E+00 GeO₂ 104.64 2.5E−06 7.5E−06 2.6E−017.8E−01 KBr 119 4.9E−07 1.5E−06 5.9E−02 1.8E−01 KI 166 5.0E−07 1.5E−068.3E−02 2.5E−01 NaF 41.99 4.9E−05 1.5E−04 2.1E+00 6.2E+00 RbCl 120.924.9E−06 1.5E−05 5.9E−01 1.8E+00 ZrOCl₂—8H₂O 178.13 4.9E−06 1.5E−058.7E−01 2.6E+00 ENERGY SUBSTRATES D-Glucose 180.16 6.9E+00 2.1E+011.2E+06 3.7E+06 Sodium Pyruvate 110.04 2.0E−01 5.9E−01 2.2E+04 6.5E+04LIPIDS Linoleic Acid 280.45 9.4E−05 2.8E−04 2.6E+01 7.9E+01 Lipoic Acid206.33 2.0E−04 7.8E−04 4.1E+01 1.6E+02 Arachidonic Acid 304.47 6.5E−061.9E−05 2.0E+00 5.9E+00 Cholesterol 386.65 5.6E−04 1.7E−03 2.2E+026.5E+02 DL-alpha 472.74 1.5E−04 4.4E−04 6.9E+01 2.1E+02tocopherol-acetate Linolenic Acid 278.43 3.5E−05 1.0E−04 9.7E+00 2.9E+01Myristic Acid 228.37 4.3E−05 1.3E−04 9.8E+00 2.9E+01 Oleic Acid 282.463.5E−05 1.0E−04 9.8E+00 2.9E+01 Palmitic Acid 256.42 3.8E−05 1.1E−049.8E+00 2.9E+01 Palmitoleic acid 254.408 3.9E−05 1.2E−04 9.8E+00 2.9E+01Stearic Acid 284.48 3.4E−05 1.0E−04 9.8E+00 2.9E+01 AMINO AClDSL-Alanine 89.09 2.5E−02 2.1E−01 2.2E+03 1.8E+04 L-Arginine 147.2 2.7E−011.5E+00 4.0E+04 2.2E+05 hydrochloride L-Asparagine-H₂O 150.13 5.0E−022.1E−01 7.5E+03 3.1E+04 L-Aspartic acid 133.1 2.5E−02 2.1E−01 3.3E+032.7E+04 L-Cysteine- 175.63 3.9E−02 1.2E−01 6.9E+03 2.1E+04 HCl—H₂OL-Cystine 313.22 3.9E−02 1.2E−01 1.2E+04 3.7E+04 dihydrochlorideL-Glutamic acid 147.13 2.5E−02 2.1E−01 3.7E+03 3.0E+04 L-Glutamine146.15 1.5E+00 4.4E+00 2.1E+05 6.4E+05 Glycine 75.07 1.5E−01 4.4E−011.1E+04 3.3E+04 L-Histidine 209.63 5.9E−02 1.8E−01 1.2E+04 3.7E+04monohydrochloride monohydrate L-Isoleucine 131.17 1.6E−01 4.9E−012.1E+04 6.4E+04 L-Leucine 131.17 1.8E−01 5.3E−01 2.3E+04 7.0E+04L-Lysine 182.65 2.0E−01 5.9E−01 3.6E+04 1.1E+05 hydrochlorideL-Methionine 149.21 4.5E−02 1.4E−01 6.8E+03 2.0E+04 L-Phenylalanine165.19 8.5E−02 2.5E−01 1.4E+04 4.2E+04 L-Proline 115.13 1.1E−01 3.2E−011.2E+04 3.7E+04 L-Serine 105.09 1.5E−01 4.4E−01 1.5E+04 4.6E+04L-Threonine 119.12 1.8E−01 5.3E−01 2.1E+04 6.3E+04 L-Tryptophan 204.231.7E−02 5.2E−02 3.5E+03 1.1E+04 L-Tyrosine 225.15 8.4E−02 3.7E−011.9E+04 8.4E+04 disodium salt hydrate L-Valine 117.15 1.8E−01 5.3E−012.1E+04 6.2E+04 VITAMINS Ascorbic acid 176.12 1.3E−01 3.8E−01 2.2E+046.7E+04 Biotin 244.31 5.6E−06 1.7E−05 1.4E+00 4.1E+00 B₁₂ 1355.372.0E−04 5.9E−04 2.7E+02 8.0E+02 Choline chloride 139.62 2.5E−02 7.5E−023.5E+03 1.1E+04 D-Calcium 238.27 1.8E−03 1.4E−02 4.4E+02 3.4E+03pantothenate Folic acid 441.4 2.4E−03 7.1E−03 1.0E+03 3.1E+03 idnositol180.16 2.7E−02 1.1E−01 4.9E+03 1.9E+04 Niacinamide 122.12 6.5E−032.0E−02 7.9E+02 2.4E+03 Pyridoxine 205.64 3.8E−03 1.1E−02 7.8E+022.4E+03 hydrochloride Riboflavin 376.36 2.3E−04 6.8E−04 8.6E+01 2.6E+02Thiamine 337.27 3.3E−03 3.6E−02 1.1E+03 1.2E+04 hydrochloride GROWTHFACTORS/ PROTEINS GABA 103.12 0 1.5E+00 0 1.5E+05 Pipecolic Acid 129 01.5E−03 0 1.9E+02 bFGF 18000 0 2.17E−07  0 3.9E+00 TGF beta 1 25000 03.5E−08 0 8.8E−01 Human Insulin 5808 0 5.9E−03 0 3.4E+04 Human Holo-78500 0 2.1E−04 0 1.6E+04 Transferrin Human Serum 67000 0 2.9E−01 02.0E+07 Albumin Glutathione 307.32 0 9.6E−03 0 2.9E+03 (reduced) OTHERCOMPONENTS Hypoxanthine Na 136.11 5.9E−03 2.6E−02 8.0E+02 3.6E+03 Phenolred 354.38 8.5E−03 2.5E−02 3.0E+03 9.0E+03 Putrescine-2HCl 161.072.0E−04 5.9E−04 3.2E+01 9.5E+01 Thymidine 242.229 5.9E−04 1.8E−031.4E+02 4.3E+02 2-mercaptoethanol 78.13 4.9E−02 1.5E−01 3.8E+03 1.1E+04Pluronic F-68 8400 1.2E−02 3.5E−02 9.8E+04 2.9E+05 Tween 80 1310 1.6E−044.9E−04 2.2E+02 6.5E+02

The liquid phase of the cell culture medium may be water, serum, oralbumin.

Many of the ingredients or components listed above in Table 1 are notnecessary, or can be used in lower concentrations.

It is contemplated that the cell culture medium may contain insulin oran insulin substitute. Similarly, the cell culture medium may containtransferrin or a transferrin substitute. However, in more specificembodiments, it is contemplated that the cell culture medium may not (1)insulin or insulin substitute, or (2) transferrin or transferrinsubstitute, or any combination of these two components.

It should be noted that other cell culture mediums may contain growthfactors such as interleukin-1 beta (IL-1β or catabolin), interleukin-6(IL6), or pigment epithelium derived factor (PEDF). Such growth factorsare not present in the cell culture medium of the present disclosure.

One specific formula for a cell culture medium is provided in Table 2:

TABLE 2 Ingredient Amount Unit bFGF 0.39 microgram (μg) Albumin 1.34milligram (mg) Insulin 2 mg Lithium Chloride 4.23 mg GABA 0.01 mg TGFbeta 1 0.06 μg Pipecolic acid 0.013 mg L-glutamine 2.92 grams MEMnon-essential amino acid solution 1 mL DMEM/F12 100 mL

In this regard, MEM non-essential amino acid solution is typicallyprovided in a 100× concentrate. The MEM of Table 2 is used afterdilution back to 1×, and contains the following amino acids in thefollowing concentration listed in Table 3:

TABLE 3 Concentration MEM Amino Acids (ng/mL) Glycine 7.50E+03 L-Alanine8.90E+03 L-Asparagine 1.32E+04 L-Aspartic acid 1.33E+04 L-Proline1.15E+04 L-Serine 1.05E+04

DMEM/F12 contains the following ingredients listed in Table 4:

TABLE 4 Concentration DMEM/F12 Ingredients (ng/mL) Glycine 187.5L-Alanine 44.5 L-Arginine hydrochloride 1475 L-Asparagine-H₂O 75L-Aspartic acid 66.5 L-Cysteine hydrochloride-H₂O 175.6 L-Cystine 2HCl312.9 L-Glutamic Acid 73.5 L-Glutamine 3650 L-Histidinehydrochloride-H₂O 314.8 L-Isoleucine 544.7 L-Leucine 590.5 L-Lysinehydrochloride 912.5 L-Methionine 172.4 L-Phenylalanine 354.8 L-Proline172.5 L-Serine 262.5 L-Threonine 534.5 L-Tryptophan 90.2 L-Tyrosinedisodium salt dihydrate 557.9 L-Valine 528.5 Biotin 0.035 Cholinechloride 89.8 D-Calcium pantothenate 22.4 Folic Acid 26.5 Niacinamide20.2 Pyridoxine hydrochloride 20 Riboflavin 2.19 Thiamine hydrochloride21.7 Vitamin B₁₂ 6.8 i-Inositol 126 Calcium Chloride (CaCl₂) (anhyd.)1166 Cupric sulfate (CuSO₄—5H₂O) 0.013 Ferric Nitrate (Fe(NO₃)₃—9H₂O)0.5 Ferric sulfate (FeSO₄—7H₂O) 4.17 Magnesium Chloride (anhydrous)286.4 Magnesium Sulfate (MgSO₄) (anhyd.) 488.4 Potassium Chloride (KCl)3118 Sodium Bicarbonate (NaHCO₃) 24380 Sodium Chloride (NaCl) 69955Sodium Phosphate dibasic 710.2 (Na₂HPO₄) anhydrous Sodium Phosphatemonobasic 625 (NaH₂PO₄—H₂O) Zinc sulfate (ZnSO₄—7H₂O) 4.32 D-Glucose(Dextrose) 31510 Hypoxanthine Na 23.9 Linoleic Acid 0.42 Lipoic Acid1.05 Phenol Red 81 Putrescine 2HCl 0.81 Sodium Pyruvate 550 Thymidine3.65

In particular, the cell culture medium may have an albumin concentrationof at least 0.3 millimolar (mM). It has been found that a 2× increase inalbumin concentration significantly improved clonal survival of humanembryonic stem cells on a laminin-521/E-Cadherin matrix. Table 5 belowprovides a formulation for a cell culture medium containing additionalalbumin.

In particular embodiments, the amount of human serum albumin (HSA) canbe varied from a concentration of 0.195 mM to 1 mM, including from 0.3mM to 1 mM or from 0.3 mM to about 0.4 mM. The amount of bFGF can alsobe varied from 0 to about 105 ng/mL, or from 0 to 3.9 ng/mL, or from 0.5ng/mL to 3.5 ng/mL. These two variations in the amount of HSA and bFGFmay occur independently or together.

TABLE 5 mTeSR1 formulation. molar Concen- Concen- mass tration trationmTeSR1 Ingredient (g/mol) (ng/mL) (mM) INORGANIC SALTS Calcium chloride(Anhydrous) 110.98 9.14E+04 8.24E−01 HEPES 238.3 2.81E+06 1.18E+01Lithium Chloride (LiCl) 42.39 4.15E+04 9.80E−01 Magnesium chloride(Anhydrous) 95.21 2.26E+04 2.37E−01 Magnesium Sulfate (MgSO₄) 120.373.84E+04 3.19E−01 Potassium chloride (KCl) 74.55 2.43E+05 3.26E+00Sodium bicarbonate (NaHCO₃) 84.01 1.51E+06 1.80E+01 Sodium chloride(NaCl) 58.44 5.53E+06 9.46E+01 Sodium phosphate, dibasic (Anhydrous)141.96 5.56E+04 3.92E−01 Sodium phosphate, monobasic 137.99 4.90E+043.55E−01 monohydrate (NaH₂PO₄—H₂O) TRACE MINERALS Ferric Nitrate(Fe(NO₃)₃—9H₂O) 404 3.92E+01 9.71E−05 Ferrous sulfate heptahydrate278.01 3.28E+02 1.18E−03 (FeSO₄—7H₂O) Copper(II) sulfate pentahydrate249.69 1.02E+00 4.08E−06 (CuSO₄—5H₂O) Zinc sulfate heptahydrate 287.563.39E+02 1.18E−03 (ZnSO₄—7H₂O) Ammonium Metavanadate NH₄VO₃ 116.981.28E+00 1.09E−05 Manganese Sulfate monohydrate 169.02 3.33E−01 1.97E−06(MnSO₄—H₂O) NiSO₄—6H₂O 262.85 2.55E−01 9.70E−07 Selenium 78.96 1.40E+011.77E−04 Sodium Meta Silicate Na₂SiO₃ 9H₂O 284.2 2.75E+02 9.66E−04 SnCl₂189.62 2.35E−01 1.24E−06 Molybdic Acid, 1235.86 2.43E+00 1.97E−06Ammonium salt CdCl₂ 183.32 2.24E+00 1.22E−05 CrCl₃ 158.36 3.14E−011.98E−06 AgNO₃ 169.87 1.67E−01 9.81E−07 AlCl₃ 6H₂O 241.43 1.18E+004.87E−06 Barium Acetate (Ba(C₂H₃O₂)₂) 255.42 2.50E+00 9.79E−06 CoCl₂6H₂O 237.93 2.33E+00 9.81E−06 GeO₂ 104.64 5.20E−01 4.97E−06 KBr 1191.18E−01 9.89E−07 KI 166 1.66E−01 1.00E−06 NaF 41.99 4.13E+00 9.83E−05RbCl 120.92 1.19E+00 9.81E−06 ZrOCl₂ 8H₂O 178.13 1.75E+00 9.80E−06ENERGY SUBSTRATES D-Glucose 180.16 2.47E+06 1.37E+01 Sodium Pyruvate110.04 4.31E+04 3.92E−01 LIPIDS Linoleic Acid 280.45 5.27E+01 1.88E−04Lipoic Acid 206.33 8.25E+01 4.00E−04 Arachidonic Acid 304.47 3.93E+001.29E−05 Cholesterol 386.65 4.33E+02 1.12E−03 DL-alphatocopherol-acetate 472.74 1.37E+02 2.90E−04 Linolenic Acid 278.431.95E+01 6.99E−05 Myristic Acid 228.37 1.96E+01 8.59E−05 Oleic Acid282.46 1.96E+01 6.94E−05 Palm itic Acid 256.42 1.96E+01 7.65E−05Palmitoleic acid 254.408 1.96E+01 7.71E−05 Stearic Acid 284.48 1.96E+016.89E−05 AMINO ACIDS L-Alanine 89.09 1.22E+04 1.37E−01 L-Argininehydrochloride 147.2 8.07E+04 5.48E−01 L-Asparagine-H₂O 150.13 2.06E+041.37E−01 L-Aspartic acid 133.1 1.82E+04 1.37E−01 L-Cysteine-HCl—H₂O175.63 1.38E+04 7.83E−02 L-Cystine dihydrochloride 313.22 2.45E+047.83E−02 L-Glutamic acid 147.13 2.02E+04 1.37E−01 L-Glutamine 146.154.30E+05 2.94E+00 Glycine 75.07 2.21E+04 2.94E−01 L-Histidinemonohydrochloride 209.63 2.47E+04 1.18E−01 monohydrate L-Isoleucine131.17 4.28E+04 3.26E−01 L-Leucine 131.17 4.64E+04 3.54E−01 L-Lysinehydrochloride 182.65 7.14E+04 3.91E−01 L-Methionine 149.21 1.35E+049.06E−02 L-Phenylalanine 165.19 2.79E+04 1.69E−01 L-Proline 115.132.49E+04 2.16E−01 L-Serine 105.09 3.09E+04 2.94E−01 L-Threonine 119.124.19E+04 3.52E−01 L-Tryptophan 204.23 7.07E+03 3.46E−02 L-Tyrosinedisodium salt hydrate 225.15 3.78E+04 1.68E−01 L-Valine 117.15 4.16E+043.55E−01 VITAMINS Ascorbic acid 176.12 4.46E+04 2.53E−01 Biotin 244.312.74E+00 1.12E−05 B12 1355.37 5.34E+02 3.94E−04 Choline chloride 139.627.02E+03 5.03E−02 D-Calcium pantothenate 238.27 8.79E+02 3.69E−03 Folicacid 441.4 2.08E+03 4.71E−03 i-Inositol 180.16 9.89E+03 5.49E−02Niacinamide 122.12 1.59E+03 1.30E−02 Pyridoxine hydrochloride 205.641.57E+03 7.62E−03 Riboflavin 376.36 1.72E+02 4.56E−04 Thiaminehydrochloride 337.27 8.16E+03 2.42E−02 GROWTH FACTORS/PROTEINS GABA103.12 1.01E+05 9.79E−01 Pipecolic Acid 129 1.27E+02 9.84E−04 bFGF 180001.04E+02 5.77E−06 TGF beta 1 25000 5.88E−01 2.35E−08 Human Insulin 58082.28E+04 3.92E−03 Human Holo-Transferrin 78500 1.08E+04 1.37E−04 HumanSerum Albumin 67000 1.31E+07 1.95E−01 Glutathione (reduced) 307.321.96E+03 6.38E−03 OTHER COMPONENTS Hypoxanthine Na 136.11 1.61E+031.18E−02 Phenol red 354.38 5.99E+03 1.69E−02 Putrescine-2HCl 161.076.36E+01 3.95E−04 Thymidine 242.229 2.86E+02 1.18E−03 2-mercaptoethanol78.13 7.66E+03 9.80E−02 Pluronic F-68 8400 1.96E+05 2.33E−02 Tween 801310 4.31E+02 3.29E−04

The systems containing a LN-521/e-cadherin substrate and mTeSR1 mediumwith additional albumin work extremely well for maintaining thedifferentiated cells in their phenotype in a completely chemicallydefined environment and xeno-free conditions without feeders or anyinhibitors of apoptosis.

It is contemplated that the cell culture medium will be completelydefined and xeno-free. The medium should also be devoid of anydifferentiation inhibitors, feeder cells, or differentiation inductors,or apoptosis inhibitors. Examples of feeder cells include mousefibroblasts or human foreskin fibroblasts. Examples of differentiationinductors include Noggin or keratinocyte growth factor.

The combination of the laminin substrate with the cell culture medium ofthe present disclosure results in a cell culture system that can becheaper, yet provides higher efficiency in maintaining differentiatedcells. In essence, all that is required is a laminin and a minimalamount of nutrition. It is particularly contemplated that the lamininused in combination with this cell culture medium is either LN-511 orLN-521.

The cell culture system in some embodiments includes at least one ofLaminin-411, Laminin-511, and Laminin-521 in the substrate, andmaintains differentiated human umbilical vein endothelial cells (HUVECs)longer than shown by conventional fibronectrin substrates.

Primary Cell Transfection

The present disclosure also relates to methods for improving thetransfection efficiency with primary cells (i.e. differentiated cells)and/or improving the survival rate of primary cells that have beentransfected.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA into which additional DNA segments may be cloned.Another type of vector is a viral vector, wherein additional DNAsegments may be cloned into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors), are integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” or simply “expression vectors”. In the presentdisclosure, the expression of the laminin polypeptide sequence isdirected by the promoter sequences of the disclosure, by operativelylinking the promoter sequences of the disclosure to the gene to beexpressed. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably, asthe plasmid is the most commonly used form of vector. However, thedisclosure is intended to include other forms of expression vectors,such as viral vectors (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses), which serve equivalentfunctions.

The vector may also contain additional sequences, such as a polylinkerfor subcloning of additional nucleic acid sequences, or apolyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the methods of the disclosure,and any such sequence may be employed, including but not limited to theSV40 and bovine growth hormone poly-A sites. Also contemplated as anelement of the vector is a termination sequence, which can serve toenhance message levels and to minimize readthrough from the constructinto other sequences. Additionally, expression vectors typically haveselectable markers, often in the form of antibiotic resistance genes,that permit selection of cells that carry these vectors.

The primary cells can be transfected using any known transfectionmethod. Such methods include a baculovirus, a lentivirus, lipofectamine,the calcium phosphate method, liposomes, cationic polymers,electroporation, sonoporation, optical transfection, geneelectrotransfer, impalefection, hydrodynamic injection, gene gun,magnetofection, and viral transduction. The vector is selected to matchwith the transfection method.

The transfected primary cells are cultured upon the substrate thatcontains the laminin. A cell culture system generally comprises asubstrate and a cell culture medium. The substrate provides a supportupon which the cells can grow. The medium provides the nutrients to thecells. It is contemplated that the cell culture medium will becompletely defined and xeno-free. The medium should also be devoid ofany differentiation inhibitors, feeder cells, or differentiationinductors, or apoptosis inhibitors. Examples of feeder cells includemouse fibroblasts or human foreskin fibroblasts. Examples ofdifferentiation inductors include Noggin or keratinocyte growth factor.

Normal transfection efficiency is about 10%. It is believed that ahigher efficiency rate can be obtained using the substrates describedherein.

The following examples are for purposes of further illustrating thepresent disclosure. The examples are merely illustrative and are notintended to limit devices made in accordance with the disclosure to thematerials, conditions, or process parameters set forth therein.

EXAMPLES A. HUVEC Cell Derivation

HUVECs were derived from umbilical cords according to a modifiedprotocol disclosed in Baudin et al., A protocol for isolation andculture of human umbilical vein endothelial cells, Nat. Protoc. 2007;2(3):481-5 (hereinafter “Baudin”). Collagenase A solution from Roche inPBS buffer was briefly injected into pre-washed vein of human umbilicalcord. Collagenase was incubated at 37 degrees Celsius, and then washedaway with cell suspension. The cell suspension was plated onlaminin-coated plates.

AI. Laminin Coating

Primary HUVECs were cultured on top of different substrate coatingsincluding conventional fibronectin as a control and recombinantlaminins, specifically human recombinant LN-411, LN-511, LN-521, LN-111,and LN-211, either alone or in combination. Combinations ofLN-411/LN-511, LN-511 alone, and LN-521 alone showed successfullong-term culture of HUVEC cells in vitro.

Laminin substrate coatings were stored in PBS at −70 degrees Celsius,were thawed on wet ice (approximately 4 degrees Celsius) and thendissolved in sterile PBS to a concentration of 5 micrograms permilliliter (ug/ml). 80 microliters (uL) of substrate solution was usedto coat 96-well plates overnight at 4 degrees Celsius or for 2 hours at37 degrees Celsius in a cell culture incubator. The wells of the 96-wellplates were pre-washed with PBS buffer prior to plating the cells.

AII. Culturing

HUVECs were cultured in sterile incubators, each with the temperatureset to 37 degrees Celsius and CO₂ levels set to 5%. Sarstedt 96-cellplates were used for culturing, with an added medium amount of 200uL/well. The medium composition used, as disclosed in Baudin, wasfiltered through a 0.22 micrometer (um) filter and stored at 4 degreesCelsius for about 2-3 weeks.

AIII. Cell Passaging

HUVEC cells were passaged in vitro at several densities. HUVEC cellswere split into 1:5, 1:10, or 1:20 splits using standard Trypsin-EDTAsolution from Gibco. Trypsin-EDTA was applied pre-warmed for about 3-5minutes. Trypsin was inhibited by serum within the cell culture medium.

AIV. HUVEC Cell Analysis

HUVECs were first characterized by quantification methods. This includedrecorded and quantifying HUVECs using the Operetta machine fromPerkin-Elmer. Magnification was chosen at 10×, 20×, and 40×.

Immunocytochemistry analysis was subsequently performed on HUVECs bypre-washing adherent cells twice with warm PBS buffer, applying 100 uLof 4% paraformaldehyde (PFA) solution, incubating at room temperaturefor 20 minutes, then washing the wells three times with PBS. Fixed cellswere permeabilized by 0.1% A Triton-X solution at room temperature for15 minutes, then washed by PBS buffer three times and blocked by 10%bovine fetal serum in PBS, supplemented by 0.1% Tween stored for 30minutes at room temperature or overnight at 4 Celsius.

HUVECs typically express the endothelial marker known as von Willebrandfactor (vWF) after 4 passages. Therefore, vWF factor was used to definepositive cells belonging to the endothelial cell type. Smooth MuscleActin (SMA) was used as a negative marker to define fibroblasts orfibroblast-like differentiated cells within in vitro population. DAPIwas used to stain the nuclei, which is necessary to define Total CellPopulation using cell population analysis on the Operetta machine usingHarmony software provided by Perkin-Elmer. Rhodamine-phalloidineconjugates were used to visualize f-actin, which acts as a marker ofcytoskeleton structure and cell borders.

Quantitative RT-PCR was performed to compare quantitative levels ofpositive marker (vWF factor) versus negative marker (SMA) in HUVECcultures in vitro after long passaging.

FIG. 3 is photomicrograph of human umbilical vein endothelial cells(HUVECs) grown on a fibronectin (FNE) substrate, 10× magnification,after 5 passages, with expression of von Willebrand factor (vWF),f-actin, and DAPI. FIG. 4 is a photomicrograph of HUVECs grown on aLN-521 substrate, 10× magnification, after 5 passages, with expressionof vWF, f-actin, and DAPI. FIG. 5 is a photomicrograph of HUVECs grownon a LN-521 substrate, 10× magnification, after 7 passages, withexpression of vWF, f-actin, and DAPI. In these three figures, the vonWillebrands factor (vWF) is stained and appears as a green color. Asseen here, the HUVECs grown on fibronectin substrate (FIG. 3) do notexpress the endothelial marker as well as cells on grown on LN-521substrate (FIG. 4 and FIG. 5), as seen by more green color expression ofvWF factor. FIG. 3 has much more black, yellow, and blue color comparedto FIG. 4 and FIG. 5, which are almost completely green. These figuresshow that LN-521 effectively prevents dedifferentiation better thanconventional fibronectin substrate. The endothelial cells grow equallywell on the fibronectin substrate, but do not maintain their phenotypeas well.

FIG. 6 is a photomicrograph of HUVECs grown on a LN-411/511 substrate,10× magnification, after 5 passages, with expression of vWF, f-actin,and DAPI. FIG. 7 is a photomicrograph of HUVECs grown on a LN-511substrate, 10× magnification, after 5 passages, with expression of vWF,f-actin, and DAPI. The vWF factor shows greater expression in cellsgrown on LN-511 relative to cells grown on LN-411/511 substrate. In FIG.6, the majority of the picture is black or yellow. In FIG. 7, there aremany more cells and each cell has some green color. However, both FIG. 6and FIG. 7 show less vWF expression than seen for cells on LN-521substrate in FIG. 4 and FIG. 5.

FIG. 8 is graph of RNA Gene Expression showing Acta2 gene expression(negative marker) and vWF gene expression (positive marker). The datawas obtained by performing qRT-PCR according to standard procedures. TheActa2 data is on the left side. The five columns are labeled, going fromleft to right, according to the table below. Note that Ln 08 refers tolaminin-411, Ln 10 refers to laminin-511, and Ln 11 refers tolaminin-521. Ln 08_10 is a mixture of laminin-411/511.

Column Text Value Left red Fibronectin 1.000 Left green Ln 08_10 0.236Left blue Ln 10 0.255 Right red Ln 11 1.640 Right green Ln 11_less_cells1.674

The vWF data is on the right side of FIG. 8. The five columns arelabeled, going from left to right, according to the table below.

Column Text Value Left red Fibronectin 1.000 Left green Ln 08_10 0.570Left blue Ln 10 1.364 Right red Ln 11 0.811 Right green Ln 11_Iess_cells0.994

FIG. 9 is a graph of quantified percentage of vWF-positive HUVECs withina population after a long-term culture of HUVECs on human recombinantlaminin-521. FIG. 10 is a graph of quantified percentage of vWF-positiveHUVECs within a population after a long-term culture of HUVECs on humanrecombinant Fibronectin. With reference to FIG. 9 and FIG. 10, thepercentage of vWF-positive HUVECs is stable after 7 passages(approximately 20.3 doublings) when Laminin-521 is used as thesubstrate. By contrast, the percentage of vWF-positive HUVECs is notstable at 5 and 7 passages when traditional fibronectin is used as thesubstrate. Therefore, recombinant LN-521 substrate enables HUVECs toretain their phenotype longer than when plated on fibronectin substrate.

FIG. 11 is a proliferation curve showing the proliferation of HUVECs ondifferent substrate coatings, dependent on days in culture up through160 days. LN-521 always had the highest number of doublings. LN-511 andLN-411/511 also supported a high number of doublings up to around 65days in culture. Around that time, their advantage over fibronectin(FNE) began to shrink.

B. Mouse Pancreatic Insulin-Producing Islet Beta Cell Derivation

Murine islet cells were derived according to the modified protocol byDong-Sheng Li et. al., as published in “A protocol for islet isolationfrom mouse pancreas,” Nature Biotechnology 2009. All manipulation withmouse pancreas was performed under a dissection microscope, with acorresponding magnification of 0.63. All instruments in contact withmouse pancreas were sterilized with ethanol solution. The bile pathwayto the duodenum in mouse subjects was blocked by clamping ampula withsurgical clamps.

A 30 gauge, one-half inch needle, was inserted into the joint site ofhepatic duct and cystic duct and inserted until reaching the middle ofcommon bile duct. Collagenase A (available from Roche) was used at aconcentration of 5 mg/ml. Collegenase A was slowly injected into murinepancreas up to volume of 3 ml and thereby inflating the pancreas.Inflated pancreas was removed and soaked in 2 ml of collagenase Asolution. Pancreas in collagenase A solution was transferred to sterile50 ml Falcon tube, incubated at temperature of 37° C. in water bath, andshaken every 5 minutes for better spreading of collagenase. After 18-25minutes of incubation in water bath, the pancreas was substantiallydissociated.

Digestion was terminated by putting the tube on ice and adding 25 ml ofice cold buffer. In order to remove exocrine cells and collagenasesolution, the islet cells were repeatedly washed and centrifuged. Theresulting suspension of cells was centrifuged at 290 g for 30 seconds at4° C. and supernatant was discarded. The remaining pellet wasresuspended with 20 ml ice-cold buffer, centrifuged again at 290 g for30 seconds at 4° C., and the supernatant was discarded. The resultingpellet was resuspended with 15 ml of buffer and poured onto a prewetted70 micrometer (μm) cell strainer. The tube was washed with 20 ml ofbuffer and poured again onto the strainer. Islet cells from the strainerwere rinsed with islet culture medium into a 100-mm tissue culture Petridish. Lastly, the islet cells were hand-picked and transferred toanother 100-mm Petri dish.

BI. Islet Beta Cell Depletion

In order to completely remove the remains of exocrine tissue andnon-islet connective tissue from islet culture, the islet beta cellswere cultured for 2-3 days in 100-mm Petri dish, which allowed the cellsand cell aggregates to settle. After that the islets were evaluated forhaving a smooth, round shape that was free from debris. Selected isletswere hand-picked and plated onto another 100-mm Petri dish.

BII. Transferring Mouse Islets onto Recombinant Human Laminin-CoatedTissue Culture-Grade Plates

Tissue-culture grade 96-well plates, e.g. available from Perkin-ElmerCell Carrier plates or Sarstedt, were coated with solutions of humanrecombinant laminins suspended in PBS solution for over 2 hours at 37 C(stored in cell incubator) or for over 20 hours at 4 C. 96-well plateswere washed 2-3 times with PBS buffer before use. Islet culture mediumwas input into the wells prior to islet plating, and was thenequilibrated in an incubator. Islet cells were hand-picked and platedonto laminin-coated plates. Culture medium was changed every 2-3 days.

BIII. Analysis of Islet Beta Cells

Islet cells were analyzed according to three different methodologies.FIGS. 12-18 pertain to analysis of beta cell morphology, which includesthe use of a phase-contrast microscope at magnification of ×4, ×10, ×20and ×40. FIG. 19 pertains to analysis of beta cell immunohistochemistry,specifically involving an antibody against C-peptide, a marker ofinsulin expression. FIG. 20-21 pertains to analysis of cellproliferation by EdU staining. The EdU molecule incorporates into DNAstrands of nuclei of cells that have divided. Rhodamine-phalloidine,used for cytoskeleton structure analysis, and Hoechst, used to stain thenuclei of cells, were also used in combination with anti-C-peptideand/or EdU for additional information.

Mouse islets were cultured on human recombinant laminins in order toimitate the natural “environmental niche” for beta cells and to enablethe beta cells to grow as a syncytium. When α5 chain laminins were usedas coatings in vitro, beta cells were able to express insulin genes. Theresult was that different types of laminins exerted different effects onmouse pancreatic islets, e.g. the ability to produce insulin orproliferate.

Laminins 411, 511, 111, and 521 were coated on a surface beforedepositing islets. FIGS. 12-16 show the results. FIG. 12 shows theislets on a surface coated with LN-521. FIG. 13 shows the islets on anuncoated surface. FIG. 14 shows the islets on a surface coated withLN-411. FIG. 15 shows the islets on a surface coated with LN-511. FIG.16 shows the islets on a surface coated with LN-111. Desirably, theislet would adhere and spread to the surface.

As seen in FIG. 12, LN-521 provided a robust, long-lasting effect ofislet adhering uniformly and spreading upon the culture plate surface.The islet of FIG. 13 did not adhere well on an uncoated surface, as seenin its generally circular shape. With reference to FIG. 14, the isletplated on LN-411 did not have a uniform shape of adhesion. The rightside adhered, but the left side failed to adhere. With reference to FIG.15, the islets plated on LN-511 had different behavior, or in otherwords were inconsistent. One islet adhered, while one did not. Lookingat FIG. 16, the islets plated on LN-111 failed to adhere and spread. Theeffect of LN-521 on islets may be due to a specific molecule interactionor characteristic.

With reference to FIGS. 17-18, a specific effect on the morphology ofthe islets is demonstrated by comparing islets deposited uponlaminin-521 in FIG. 17 to islets deposited upon laminin-111 in FIG. 18.Mouse pancreatic insulin-producing beta islets expanded on humanrecombinant laminin-521, whereas islets expanded on laminin-111 retainedtheir three-dimensional spherical shape. Differences in morphology andin the cell community infrastructure can be seen on either lowmagnification (×10) (FIGS. 17(a) and 18(a)) or high magnification (×40)(FIGS. 17(b) and 18(b)).

With reference to FIG. 19, after 3-4 weeks in culture, beta islets weredeposited upon laminin-521 and stained positively for C-peptide, whichis a marker for insulin expression. FIG. 19(a) shows a positiveindication of C-peptide, and therefore successful production of insulin,when beta islets are deposited on laminin-521. FIG. 19(b) shows thepositive indication of C-peptide, as well as the nucleus of beta isletson laminin-521, shown through blue colored Hoechst indicator (the upperright dot).

With reference to FIG. 20, after 3-4 weeks in culture, beta isletsdeposited on laminin-521 maintained capacity for proliferation. Withreference to FIG. 20(a), beta islets demonstrated positive EdU staining,shown by a green color (the solid dots) in the nuclei of proliferatedcells. The nuclei are spread out relative to the beta islets in FIG. 19.In FIG. 20(b), the green color EdU stain was merged with aphase-contrast photograph for additional context on the location ofproliferated nuclei.

In FIG. 21, the joint effect of proliferation of islets cultured uponlaminin-521 and expression of C-peptide within the same islets isdemonstrated. With reference to FIG. 21(a), mouse pancreaticinsulin-producing beta islet cells when cultured on human recombinantlaminin-521 maintained proliferation potential and expressed C-peptide.This was demonstrated by the contemporaneous positive green EdU stainingas well as the orange color (indication of c-peptide, marker ofinsulation production) in FIG. 21(a). The green EdU stains were thesharp bright points, while the orange color was the portions surroundingthe sharp points. In FIG. 21(b), blue Hoechst dye was localized in thenuclei of all islets, while green EdU indicated the nuclei of isletsdivided during the last three days. The islets in FIG. 21(b) that havedivided during the last three days are farther spaced apart than thenuclei of all islet cells shown by Hoechst. The blue nuclei were theextremely bright spots, while the green nuclei were the less brightspots around the perimeter.

The present disclosure has been described with reference to exemplaryembodiments. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the present disclosure be construed asincluding all such modifications and alterations insofar that they comewithin the scope of the appended claims or the equivalents thereof.

1. A method for maintaining the phenotype of a differentiated cell, comprising: plating the differentiated cell on a cell culture substrate comprising a laminin, wherein the laminin is an intact protein or a protein fragment.
 2. The method of claim 1, wherein the differentiated cell is an endothelial cell, a cardiomyocyte, a dopamine producing cell, a hepatocyte, a pancreatic islet, or a pancreatic beta cell.
 3. The method of claim 1, wherein the laminin is laminin-521 or laminin-511.
 4. The method of claim 1, wherein the laminin is an effective recombinant laminin.
 5. The method of claim 1, wherein the cell culture substrate further comprises a cadherin.
 6. The method of claim 5, wherein the cadherin is e-cadherin.
 7. The method of claim 5, wherein the weight ratio of the laminin to the cadherin is from about 5:1 to about 15:1.
 8. The method of claim 5, wherein the weight ratio of the laminin to the cadherin is from about 5:1 to about 10:1.
 9. The method of claim 5, wherein the laminin is laminin-521 and the cadherin is e-cadherin.
 10. The method of claim 5, wherein the cell culture substrate consists of the laminin and the cadherin.
 11. The method of claim 1, wherein the cell culture substrate does not contain any differentiation inhibitors, feeder cells, differentiation inductors, or apoptosis inhibitors.
 12. The method of claim 1, further including applying a cell culture medium to the first stem cell.
 13. The method of claim 12, wherein the cell culture medium has an albumin concentration of at least 0.3 mM.
 14. A cell-based assay system, comprising: a substrate comprising a laminin, wherein the laminin is an intact protein or a protein fragment; and differentiated cells plated upon the substrate.
 15. A method of improving the transfection efficiency of differentiated cells, comprising: plating differentiated cells on a substrate containing a laminin, wherein the laminin is an intact protein or a protein fragment; transfecting the differentiated cells with a vector; and culturing the transfected cells.
 16. The method of claim 15, wherein the laminin is laminin-521 or laminin-511.
 17. The method of claim 15, wherein the substrate further comprises a cadherin.
 18. The method of claim 17, wherein the cadherin is e-cadherin.
 19. The method of claim 17, wherein the weight ratio of the laminin to the cadherin is from about 5:1 to about 15:1.
 20. The method of claim 15, wherein the differentiated cells are transfected using a baculovirus, a lentivirus, lipofectamine, the calcium phosphate method, liposomes, cationic polymers, electroporation, sonoporation, optical transfection, gene electrotransfer, impalefection, hydrodynamic injection, gene gun, magnetofection, and viral transduction. 